PROJECT SUMMARY The immune system plays a critical role in human health by sensing and destroying foreign invaders such as viruses, bacterium, parasites, and other sources of infection. Activating the immune response can help fight off a range of diseases such as malaria, tuberculosis, and cancer. Conversely, inhibiting the immune response can help treat autoimmune diseases such as diabetes, arthritis, and atherosclerosis. As such, there has been significant and growing interest in developing new therapeutics for regulating the immune response, both in stimulating or suppressing it, to treat a wide variety of human diseases. One particular area of interest lies in the development of Toll-like receptor (TLR) agonists. TLRs are a well-studied family of pattern recognition receptors that stimulate the innate immune response when activated by the appropriate agonist. However, most TLR agonist-based therapeutics are not selective for which physiological regions are immunologically stimulated. TLR activity and the ensuing immune response may thus occur indiscriminately, leading to inflammation responses at undesired physiological sites, which can lead to disease exacerbation. Synthetically engineering the TLR agonist so that its primary release occurs specifically at disease sites may potentially increase immunotherapeutic potency while also reducing unwanted inflammation at healthy locations. The driving hypothesis of this proposal is to determine whether TLR agonists can be caged so that they are immunologically inactive until they detect specific bioanalytes, at which point they will self-immolate to release the free agonists and stimulate the immune response. The ability to cage and release TLR agonists was demonstrated previously by Esser-Kahn and coworkers, who utilized directed UV light to release TLR agonists and stimulate innate immune cells. We will use similar tools and metrics (NF-?B activity, cytokine and cell surface marker production, confocal microscopy) to characterize immune cell activation here, but exploit the local chemistry at the disease site to achieve higher specificity over uncaged therapeutics. The specific aims of this proposal that we will use to validate our hypothesis are to establish cage-and-release systems that are triggered by two specific bioanalytes: (a) the ferrous ion (Fe2+), and (b) hydrogen peroxide (H2O2). These bioanalytes were selected under the guiding principle that cancer cells contain severely elevated levels of both these species. However, our therapeutic strategy should be applicable to other disease states that involve dysregulated iron metabolism or generate high levels of H2O2. We will study the immune response in RAW- blue 264.7 macrophage reporters and primary dendritic cells that have been exposed to varying levels of Fe2+ and H2O2. Upon detecting these bioanalytes, the cage will fall apart and the immunotherapeutic payload will be released. If our hypothesis is validated, our findings may establish TLR agonists as therapeutics that can initiate immune responses at specific locations or under specific disease conditions (high levels of Fe2+ or H2O2), leading to enhanced cell-mediated immunity and enabling more potent and directed therapeutics.